Steering CAR T cell epigenetic programs by tweaking manufacturing protocol
Authors: Meghan W. Dukes, Giedre Krenciute
Journal: Cell Reports Medicine (2023)

CAR T cell persistence remains a significant roadblock to effective clinical translation of CAR T cells for solid and brain tumors. Jung et al.1Jung I.Y. Noguera-Ortega E. Bartoszek R. Collins S.M. Williams E. Davis M. Jadlowsky J.K. Plesa G. Siegel D.L. Chew A. et al.Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors.Cell Rep. Med. 2023; 4: 101053https://doi.org/10.1016/j.xcrm.2023.101053Abstract Full Text Full Text PDF Google Scholar demonstrate enrichment of resident memory phenotypes through simple changes to the CAR T cell manufacturing process. CAR T cell persistence remains a significant roadblock to effective clinical translation of CAR T cells for solid and brain tumors. Jung et al.1Jung I.Y. Noguera-Ortega E. Bartoszek R. Collins S.M. Williams E. Davis M. Jadlowsky J.K. Plesa G. Siegel D.L. Chew A. et al.Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors.Cell Rep. Med. 2023; 4: 101053https://doi.org/10.1016/j.xcrm.2023.101053Abstract Full Text Full Text PDF Google Scholar demonstrate enrichment of resident memory phenotypes through simple changes to the CAR T cell manufacturing process. Chimeric antigen receptor (CAR) T cells are gaining momentum as safe treatments of solid and brain tumors; however, improving anti-tumor efficacy remains a challenge for the field. The mechanism by which CAR T cells kill tumor cells is straightforward. Synthetic CAR molecules designed to recognize specific tumor antigens redirect T cell cytotoxic machinery to antigen-expressing tumor cells, which CAR T cells recognize and kill. CAR T cells subsequently activate and expand, promoting further tumor cell killing. However, it is well established that CAR T cells progressively lose effector function after repeat exposure to tumor cells, resulting in incomplete tumor clearance and recurrence. This gradual CAR T cell functional loss and inability to expand and persist are collectively defined as T cell exhaustion. To overcome exhaustion, the field is exploring strategies for manipulating CAR T cell functional programs so that effector function is sustained long term. Targeting T cell epigenetic programs has emerged as a potential way to accomplish this. Indeed, several groups have explored methods of re-programming the long-term effector function of CAR T cells by targeting epigenetic programs. In human CAR T cells, deletion of TET2,2Jain N. Zhao Z. Feucht J. Koche R. Iyer A. Dobrin A. Mansilla-Soto J. Yang J. Zhan Y. Lopez M. et al.TET2 guards against unchecked BATF3-induced CAR T cell expansion.Nature. 2023; 615: 315-322Crossref PubMed Scopus (0) Google Scholar DNMT3A,3Prinzing B. Zebley C.C. Petersen C.T. Fan Y. Anido A.A. Yi Z. Nguyen P. Houke H. Bell M. Haydar D. et al.Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity.Sci. Transl. Med. 2021; 13eabh0272Crossref Scopus (56) Google Scholar or SUV39H14Jain N. Zhao Z. Koche R. Gozlan Y. Brocks D. Raveh-Sadka T. Wells D. Dobrin A. Shi Y. Lopez M. et al.Abstract 5583: SUV39H1 disruption enhances the persistence and anti-tumor efficacy of CD28-costimulated human CAR T cells.Cancer Res. 2022; 82: 5583Crossref Google Scholar significantly enhances CAR T cell survival, proliferation, and effector function. However, in these examples, improving CAR T cell function requires deletion of epigenetic regulators via gene editing of CAR T cells. While gene knockout approaches are now well established in the CAR T cell field, translation of these approaches to clinic is still highly tedious, time consuming, and expensive due to regulatory requirements. Thus, alternative and simplistic approaches to overcome T cell exhaustion are needed. One strategy that has been explored is tweaking the CAR T cell manufacturing protocol through the addition of chemical reagents. Multiple studies have demonstrated that addition of dasatinib during CAR T cell manufacturing not only primes CAR T cells to adopt a memory-like phenotype, reprograms transcriptional and epigenetic profiles, and prevents exhaustion in CAR T cells in vitro and in vivo,5Weber, E. W.; Parker, K. R.; Sotillo, E.; Lynn, R. C.; Anbunathan, H.; Lattin, J.; Good, Z.; Belk, J. A.; Daniel, B.; Klysz, D., et al, Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science (New York, N.Y.) 2021, 372.Google Scholar but also overcomes T cell fratricide.6Watanabe N. Mo F. Zheng R. Ma R. Bray V.C. van Leeuwen D.G. Sritabal-Ramirez J. Hu H. Wang S. Mehta B. et al.Feasibility and preclinical efficacy of CD7-unedited CD7 CAR T cells for T cell malignancies.Mol. Ther. 2023; 31: 24-34Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar Addition of decitabine, an established DNA methyltransferase (DNMT) inhibitor, also shows promise in priming CAR T cells for improved effector function.7Wang Y. Li M. Chen L. Bian H. Chen X. Zheng H. Yang P. Chen Q. Xu H. Liu Z. et al.Low-dose decitabine priming endows CAR T cells with enhanced and persistent antitumour potential via epigenetic reprogramming.Nat. Commun. 2021; 6: 409Crossref Scopus (75) Google Scholar In a current study published by Cell Reports Medicine, Jung. et al.1Jung I.Y. Noguera-Ortega E. Bartoszek R. Collins S.M. Williams E. Davis M. Jadlowsky J.K. Plesa G. Siegel D.L. Chew A. et al.Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors.Cell Rep. Med. 2023; 4: 101053https://doi.org/10.1016/j.xcrm.2023.101053Abstract Full Text Full Text PDF Google Scholar developed a method by which CAR T cell products can be enriched for tissue-resident memory (TRM) phenotypes to encourage CAR T cell accumulation in tumor tissue. While many blood cancers benefit from CAR T cells that actively circulate through the bloodstream and bone marrow, effective treatment of solid tumors requires cellular programming that encourages cytolytic CAR T cells to persist at the location of a tumor and resist recirculation. To accomplish this, Jung et al. optimized a CAR T cell production protocol in which T cells are activated and CAR products are generated in the presence of exogenous transforming growth factor β (TGF-β) (Figure 1). TGF-β has numerous functions on T cells, including maintenance of naive/stem-like T cell populations and implication in preserved T cell tissue retention,8Mackay L.K. Rahimpour A. Ma J.Z. Collins N. Stock A.T. Hafon M.L. Vega-Ramos J. Lauzurica P. Mueller S.N. Stefanovic T. et al.The developmental pathway for CD103(+)CD8+ tissue-resident memory T cells of skin.Nat. Immunol. 2013; 14: 1294-1301Crossref PubMed Scopus (824) Google Scholar but also reduction of CD8+ T cell cytolytic effector function.9Oh S.A. Li M.O. TGF-β: guardian of T cell function.J. Immunol. 2013; 191 (1950): 3973-3979Crossref PubMed Scopus (202) Google Scholar Mesothelin (M5)-targeted CAR T cells generated in the presence of interleukin-2 (IL-2) and TGF-β (CAR-TRM cells) were analyzed for cell surface markers (via flow cytometry), transcriptional profiles (via bulk and single-cell RNA sequencing), and chromatin accessibility (via assay for transposase-accessible chromatin using sequencing [ATAC-seq]) to confirm enrichment of CAR-TRM phenotypes. CAR T cells generated in the presence of TGF-β indeed showed increased surface expression and transcriptional regulation of TRM phenotype compared with CAR T cells generated in IL-2 alone. Importantly, transcriptional analysis revealed downregulation of the KLF2 gene loci that dictates T cell commitment to recirculation, suggesting CAR-TRM cells are enriched for gene profiles that encourage tissue residence and discourage circulation. CAR-TRM cells also showed increased expression of markers associated with T cell stemness, self-renewal, and early memory as well as increased expression of traditional exhaustion markers. However, CAR-TRM cells showed superior cytokine production upon repeat stimulation with tumor antigens compared with control CAR T cells and reduced expression of genes responsible for terminal differentiation and dysfunction. This suggests that the exhaustive phenotypes of CAR-TRM cells are decoupled from anti-tumor efficacy and the CAR-TRM cells are not terminally exhausted. Indeed, CAR-TRM cells produced with exogenous TGF-β showed superior in vivo anti-tumor efficacy compared with control CAR T cells in models of both solid (pancreatic and prostate) and liquid (B cell leukemia) cancers. Specifically, CAR-TRM administration resulted in significantly higher overall number of resident memory phenotype CAR T cells in tumor tissues two weeks following treatment. Improved accumulation of CAR-TRM cells in tumor tissues was further observed as early as two days following administration. Importantly, animals that received CAR-TRM cells successfully cleared tumor rechallenges, suggesting the improved tissue residency and memory of these cells. Due to the known T cell inhibitory functions of TGF-β on T cells, Jung et al. attempted to enrich for TRM phenotypes by overexpressing RUNX3 in CAR T cells. While these cells showed superior activation via expression of CD25, they ultimately failed to induce TRM phenotypes. While the study is exciting and shows promising results, there are some questions that remain to be addressed. For example, this study was only performed in solid tumor and B cell leukemia settings. It would be interesting to see whether results are consistent in brain tumors and more challenging hematological malignancies such as acute myeloid leukemia. In addition, multiple published studies try to block TGF-β signaling because of its immunosuppressive effect on CAR T cells.10de Folmont A. Bourhis J.-H. Chouaib S. Terry S. Multifaceted Role of the Transforming Growth Factor β on effector T cells and the implication for CAR-T cell Therapy.Immuno. 2021; 1: 160-173Crossref Google Scholar Thus, it would be interesting to know whether TGF-β priming of CAR T cells can overcome observed TGF-β suppressive effects of the TME where it is secreted by infiltrating immune cells. For this, studies using immune-competent animal models are warranted. In summary, the study by Jung et al.1Jung I.Y. Noguera-Ortega E. Bartoszek R. Collins S.M. Williams E. Davis M. Jadlowsky J.K. Plesa G. Siegel D.L. Chew A. et al.Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors.Cell Rep. Med. 2023; 4: 101053https://doi.org/10.1016/j.xcrm.2023.101053Abstract Full Text Full Text PDF Google Scholar demonstrates that small tweaks in the CAR T cell manufacturing protocol can generate TRM CAR T cells with enhanced ability to penetrate solid tumors and resist functional exhaustion. While additional pre-clinical testing is warranted, CAR T cell priming with TGF-β has shown promise to easily manipulate CAR T cell functionality with potentially easy translation to the clinic. G.K. was a one-time consultant for Kineticos (2022).